Antigen Retrieval Protocol (PIER)

Fixing proteins with formalin creates crosslinks. In the process of crosslinking, antigen in the tissue can be masked, restricting antibody-target binding. The impaired ability of the antibody to access its epitope can result in weak signal or false negative staining. To promote epitope availability and enhance immunogenicity, one of several antigen retrieval methods is used. Proteolytic-Induced Epitope Retrieval (PIER) is an enzymatic method of antigen retrieval which relies on enzymes such as proteinase K, trypsin, or pepsin to unmask antigen. Heat-Induced Epitope Retrieval (HIER) utilizes heat to promote epitope availability. Optimal retrieval conditions depend on the type of tissue, fixation, and antibody, necessitating optimization for each antigen.

Proteolytic-Induced Epitope Retrieval (PIER)

Trypsin Working Solution (0.05%):

Trypsin stock solution (0.5%) --------------- 1 ml

Calcium chloride stock solution 1% ------ 1 ml

Distilled Water ------------------------------------ 8 ml

Adjust pH to 7.8 with 1N NaOH.

  1. Cover sections with trypsin working solution.
  2. Incubate for 10-20 minutes at 37 degrees Celsius in humidified chamber
  3. Allow sections to cool at room temperature for 10 minutes.

Note: Optimal incubation time will vary depending on tissue type and degree of fixation. Optimal conditions should be determined by the user.

Proteinase K Working Solution (1x, 20 ug/ml):

Proteinase K Stock Solution (20X) ------ 1 ml

TE Buffer, pH8.0 ------------------------------- 19 ml

Mix well.

  1. Cover sections with Proteinase K working solution.
  2. Incubate for 10-20 minutes at 37 degrees Celsius in humidified chamber
  3. Allow sections to cool at room temperature for 10 minutes.

Note: Optimal incubation time will vary depending on tissue type and degree of fixation. Optimal conditions should be determined by the user.

Proteinase K Reagents